MCM7 Antibody

Basic Research Questions

• What is MCM7 and what is its biological significance in cellular processes?

MCM7 is a 719 amino acid protein belonging to the MCM family that acts as a component of the MCM2-7 complex, which is the putative replicative helicase essential for "once per cell cycle" DNA replication initiation and elongation in eukaryotic cells. The MCM2-7 complex participates in pre-replication complex formation and exhibits helicase activity that unwinds DNA, resulting in recruitment of DNA polymerases and initiation of DNA replication .

MCM7 plays a crucial role in regulating DNA replication, ensuring DNA is replicated only once per cell cycle, thus maintaining genomic stability and preventing over-replication that can lead to genomic instability and cancer . Recent structural analyses have revealed that conserved loop structures located inside the ring structure of the MCM complex interact with unwound single-stranded DNA to facilitate translocation along the DNA during replication .

• What applications are MCM7 antibodies validated for in research settings?

Based on the search results, MCM7 antibodies have been validated for multiple research applications:

These applications enable researchers to investigate MCM7 expression, localization, and interactions in various experimental models .

• What species reactivity do commonly available MCM7 antibodies demonstrate?

Commercial MCM7 antibodies have been validated for reactivity with multiple species:

This cross-species reactivity makes these antibodies valuable tools for comparative studies across different model organisms . When selecting an antibody for your experimental system, consider both the species reactivity and the specific application requirements to ensure optimal results.

• How can researchers optimize sample preparation for MCM7 detection in different applications?

Sample preparation optimization is critical for successful MCM7 detection:

For Western Blot:

• Use RIPA or NP-40 lysis buffers with protease inhibitors

• Process samples under reducing conditions

• Load 20-50 μg of total protein per lane

• Validated cell lines include HeLa, MCF-7, NIH/3T3, Jurkat, K-562, and Daudi

For Immunohistochemistry:

• Test multiple antigen retrieval methods:

  • TE buffer pH 9.0 (recommended)

  • Citrate buffer pH 6.0 (alternative)

• MCM7 antibodies have been validated on human lung squamous cell carcinoma, lung cancer, lymphoma, and mesothelioma tissues

For Immunofluorescence:

• Fix cells with 4% paraformaldehyde (10 minutes at room temperature)

• Permeabilize with 0.1% Triton X-100

• Include a blocking step with 1-5% BSA or normal serum

• A431 or HeLa cells serve as good positive controls

For Flow Cytometry:

• Ensure proper fixation and permeabilization for intracellular staining

• Use 0.40 μg antibody per 10^6 cells in 100 μl suspension

• Include appropriate isotype controls

• HepG2 cells are recommended as positive controls

• What controls should be included when using MCM7 antibodies in experimental procedures?

Proper controls are essential for interpretation of results with MCM7 antibodies:

Positive Controls:

• Western blot: HeLa, MCF-7, Jurkat, or K-562 cell lysates (expect ~81 kDa band)

• IHC: Human lung cancer tissue or mesothelioma tissue (nuclear staining)

• IF/ICC: A431 or HeLa cells (nuclear localization)

• Flow cytometry: HepG2 cells

Negative Controls:

• Primary antibody omission control

• Isotype-matched control antibody

• MCM7-knockdown/knockout samples (if available)

• Non-expressing tissues or cell types

Validation Controls:

• Peptide competition assay to confirm specificity

• Detection with secondary antibody matching host species (rabbit or mouse IgG)

• Multiple MCM7 antibodies targeting different epitopes

• Correlation with MCM7 mRNA expression data

Including these controls helps ensure specificity of detection and allows proper interpretation of MCM7 expression patterns across experimental conditions.

Advanced Research Questions

• How does MCM7 function within the MCM2-7 complex during DNA replication, and what techniques can be used to study this process?

MCM7 is one of six proteins (MCM2-7) that form a heterohexameric complex functioning as the replicative DNA helicase during DNA replication. Recent structural analyses have revealed that conserved loop structures located inside the ring structure of the MCM2-7 complex interact with unwound single-stranded DNA to allow the complex to translocate along the DNA .

To effectively study MCM7 function within this complex, researchers can employ:

a) ChIP (Chromatin Immunoprecipitation) assays:

• Determine MCM7 binding to specific DNA regions

• Monitor temporal association with replication origins

• Study how stimulation with agents like synthetic androgen R1881 affects MCM7 binding to genomic DNA

b) Co-immunoprecipitation with MCM7 antibodies:

• Identify interactions with other MCM proteins and replication factors

• Study dynamic assembly/disassembly of the MCM2-7 complex

• Investigate regulatory interactions such as those with androgen receptor

c) DNA fiber analysis combined with MCM7 immunofluorescence:

• Track replication fork progression

• Identify origin firing events

• Correlate with MCM7 localization and abundance

d) Cell cycle synchronization with MCM7 immunodetection:

• Monitor temporal and spatial regulation of MCM7 during different cell cycle phases

• Analyze effects of replication stress on MCM7 localization and function

e) Proximity ligation assays:

• Detect in situ interactions between MCM7 and other replication factors

• Quantify interaction dynamics throughout S-phase

• What is the connection between MCM7 expression and cancer progression, and how can researchers effectively study this relationship?

Mechanistic insights into MCM7's oncogenic role include:

• Knockdown of MCM7 significantly inhibits cellular proliferation in vitro and HCC tumorigenicity in vivo

• MCM7 regulates cyclin D1 through the MAPK signaling pathway

• High expression of both MCM7 and cyclin D1 exhibits relatively high sensitivity and specificity in predicting worse outcomes for HCC patients

Methodology for studying MCM7 in cancer research:

a) Tissue microarray analysis:

• Use MCM7 antibodies at 1:200-1:800 dilution

• Perform antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0)

• Correlate with patient outcome data and clinical parameters

b) Gene expression manipulation:

• siRNA or shRNA-mediated knockdown of MCM7

• CRISPR/Cas9-based gene editing

• Rescue experiments with wild-type or mutant MCM7

c) Signaling pathway analysis:

• Western blot analysis of MAPK pathway components

• Co-expression analysis of MCM7 and cyclin D1

• Pharmacological manipulation of signaling pathways

d) In vivo models:

• Xenograft models with MCM7-modified cancer cells

• Analysis of tumor growth, invasion, and metastasis

• Immunohistochemical validation in tumors

• How does MCM7 interact with androgen receptor (AR), and what implications does this have for prostate cancer research?

Research has revealed that MCM7 interacts with androgen receptor (AR) with high affinity both in vitro and in vivo . This interaction has significant implications for prostate cancer research, given that MCM7 is both amplified and overexpressed in metastatic prostate cancer.

Key findings about the MCM7-AR interaction:

• The AR-binding motif for MCM7 comprises amino acids 221 to 248

• The MCM7-binding motif for AR comprises amino acids 426 to 475

• AR stimulation with high doses of synthetic androgen R1881 leads to:

  • Decreased MCM7 binding to genomic DNA

  • Reduction in DNA synthesis

  • Decreased number of cells progressing through S phase

Methods to study this interaction in prostate cancer research:

a) Co-immunoprecipitation assays:

• Use MCM7 antibodies to pull down AR complexes or vice versa

• Analyze interaction dynamics in response to hormonal stimulation

• Examine changes in complex formation during disease progression

b) Deletion/mutation analysis:

• Create mutants lacking the specific interaction motifs

• Express these constructs in prostate cancer cell lines

• Assess effects on AR signaling, DNA replication, and cell proliferation

c) ChIP-seq analysis:

• Identify genomic regions where MCM7 and AR co-localize

• Analyze effects of androgen stimulation on co-occupancy patterns

• Correlate with transcriptional and replication regulatory regions

d) Cell cycle analysis in AR-positive versus AR-negative prostate cancer models:

• Compare MCM7 expression and localization patterns

• Assess sensitivity to androgen stimulation

• Evaluate impact on DNA replication and cellular proliferation

• What is the role of MCM7 polyubiquitylation in DNA replication termination, and how can this process be investigated?

The search results indicate that polyubiquitylation of MCM7 is involved in terminating MCM function during DNA replication . This post-translational modification represents a critical regulatory mechanism that ensures proper completion of DNA replication and prevents re-replication.

Methodological approaches to study MCM7 polyubiquitylation:

a) Biochemical detection:

• Immunoprecipitation of MCM7 followed by Western blotting with ubiquitin antibodies

• Use of proteasome inhibitors (e.g., MG132) to stabilize ubiquitylated species

• Analysis of ubiquitin chain types (K48 vs. K63) to determine functional outcomes

b) Identification of ubiquitylation sites:

• Mass spectrometry analysis of immunoprecipitated MCM7

• Site-directed mutagenesis of lysine residues

• Functional analysis of ubiquitylation-resistant MCM7 mutants

c) Cell cycle-dependent regulation:

• Synchronization of cells at different stages of S phase

• Time-course analysis of MCM7 ubiquitylation during replication

• Correlation with replication termination events

d) Functional consequences:

• Analysis of replication termination in cells expressing ubiquitylation-resistant MCM7

• DNA fiber assays to monitor replication fork progression and termination

• Assessment of genomic instability resulting from defective termination

• How can researchers resolve discrepancies in MCM7 expression or localization data across different experimental systems?

When researchers encounter conflicting data regarding MCM7 expression or localization, several methodological approaches can help resolve these discrepancies:

a) Antibody validation:

• Confirm specificity using genetic knockdown/knockout controls

• Test multiple antibodies targeting different epitopes of MCM7

• Verify consistent detection at the expected molecular weight (81 kDa)

b) Cell cycle considerations:

• MCM7 expression and localization vary throughout the cell cycle

• Synchronize cells or use cell cycle markers to properly interpret results

• Consider that MCM7 gene expression is regulated during cellular aging and the cell cycle

c) Experimental conditions:

• Standardize fixation and permeabilization protocols

• Control for oxygen concentration, which affects MCM7 expression

• Consider the impact of growth conditions and cell density

d) Detection methods:

• Use complementary approaches (e.g., IF and biochemical fractionation)

• Apply quantitative methods when possible

• Include appropriate positive and negative controls

e) Model system differences:

• Compare results across cell lines, primary cells, and tissue samples

• Consider species-specific differences in MCM7 regulation

• Validate findings in physiologically relevant systems

f) Pathological states:

• Account for altered MCM7 regulation in disease states

• Consider genomic alterations affecting MCM7 in cancer cells

• Compare normal versus transformed cells from the same tissue origin

• How do mutations in MCM7 contribute to genomic instability and what methodologies can detect their functional consequences?

The search results indicate that disruptions of MCM2-7 function can lead to genomic instability and cancer progression . A point mutation in MCM4 that disturbs MCM2-7 complex function results in genomic instability, leading to cancer cell generation . Similarly, mutations in MCM7 would likely have comparable effects given its critical role in the MCM2-7 complex.

Methodological approaches to study the impact of MCM7 mutations:

a) Mutation identification and characterization:

• Next-generation sequencing of MCM7 in cancer samples

• Functional domain mapping of identified mutations

• Structural modeling to predict effects on MCM2-7 complex assembly

b) Genomic instability assays:

• Micronucleus formation assays

• Chromosomal aberration analysis

• DNA damage response activation (γH2AX foci)

• Sister chromatid exchange frequency

c) Replication stress analysis:

• DNA fiber assays to measure replication fork speed and stalling

• Analysis of dormant origin firing patterns

• RPA and RAD51 foci formation as indicators of replication stress

d) MCM7 complex function:

• Helicase activity assays with purified components

• MCM2-7 complex formation analysis

• Single-molecule studies of DNA unwinding

e) Cell-based functional studies:

• CRISPR/Cas9 knock-in of specific MCM7 mutations

• Analysis of cell cycle progression and checkpoint activation

• Long-term genomic stability assessment in culture

• What are the most effective approaches for studying the cell cycle-dependent regulation of MCM7?

MCM7 expression and localization change throughout the cell cycle, reflecting its critical role in DNA replication. Several methodological approaches can effectively study these cell cycle-dependent changes:

a) Cell synchronization techniques:

• Double thymidine block for G1/S boundary synchronization

• Nocodazole treatment for mitotic arrest

• Serum starvation for G0/G1 accumulation

• Release time-course sampling to follow progression through the cycle

b) Flow cytometry analysis:

• Co-staining for MCM7 (using validated antibodies at 0.40 μg per 10^6 cells)

• DNA content measurement with propidium iodide or DAPI

• EdU incorporation to identify S-phase cells

• Quantification of MCM7 levels across cell cycle phases

c) Chromatin fractionation combined with Western blotting:

• Separation of soluble and chromatin-bound protein fractions

• Western blot analysis of MCM7 distribution (1:5000-1:50000 dilution)

• Cell cycle-dependent changes in chromatin association

d) Immunofluorescence microscopy:

• Fixed-cell analysis at different cell cycle stages

• Co-staining with cell cycle markers

• Quantitative image analysis of nuclear vs. cytoplasmic MCM7

• Recommended antibody dilution: 1:50-1:500 for IF/ICC

e) Live-cell imaging:

• Fluorescently-tagged MCM7 constructs

• Time-lapse microscopy to track dynamic changes

• Co-imaging with cell cycle phase markers

• How can researchers effectively investigate the interplay between MCM7 and other proteins in the replication machinery?

Understanding how MCM7 interacts with other components of the replication machinery is crucial for elucidating its function. Several methodological approaches are effective for studying these interactions:

a) Co-immunoprecipitation assays:

• Use MCM7 antibodies (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate)

• Identify interaction partners by mass spectrometry

• Verify specific interactions by Western blotting

• Include appropriate controls (IgG, input samples)

b) Proximity ligation assays (PLA):

• Detect protein-protein interactions in situ

• Quantify interaction frequency in different cell compartments

• Compare interaction patterns throughout cell cycle

c) ChIP-seq and ChIP-reChIP:

• Map genome-wide MCM7 binding sites

• Identify co-localization with other replication factors

• Determine sequential or simultaneous binding events

d) Bimolecular fluorescence complementation (BiFC):

• Visualize interactions between MCM7 and other proteins in living cells

• Track dynamic formation of protein complexes

• Map domains required for interaction

e) FRET/FLIM analysis:

• Measure direct protein-protein interactions

• Determine interaction distances

• Analyze interaction dynamics in real-time

f) Protein complex reconstitution:

• In vitro assembly of MCM2-7 complex

• Functional assays with purified components

• Structure-function analysis of replication complexes

These methodologies provide complementary approaches to understand how MCM7 functions within the broader context of the DNA replication machinery, allowing researchers to dissect both stable and transient interactions that regulate this complex process.